رضا فلک

The pattern of incidence of asthma varies with age and sex, as females suffer more than males. Some asthmatic women report premenstrual exacerbation of asthma symptoms as well as variation of its severity during pregnancy, thus it is believed that sex hormonal changes could affect asthma. Hormone Replacement Therapy (HRT) is a routine and accepted procedure which is used for treatment of several cases such as irregular periods, hirsutism, menopausal manifestations, acne, osteoporosis and amelioration of the symptoms in some autoimmune disease. HRT could reduce the magnitude of variations in estrogen and progesterone over the menstrual cycle. According to increased asthma prevalence among women than men and regarding to expression of estrogen and progesterone receptor on lung and immune cells, we aimed to determine the effects of 17β-estradiol (E2) and progesterone (P4) alone and in combination form on expression of T-bet and IFN-ɣ cytokine secretion, in correlation with Th1 cell subset of Peripheral Blood Mononuclear Cells (PBMC) (as crucial cells that could affect cytokines’ balance) in asthmatic patients versus non-asthmatic healthy controls.
The diagnosis of asthma was confirmed on the basis of clinical symptoms and detection of allergen specific IgE. Then PBMCs were isolated and cultivated in 24-well plates in the presence or absence of 1% phytohemagglutinin (PHA), 10-8 M of estrogen and 10-6 M of progesterone, followed by mRNA isolation. After reverse transcription, real-time quantitative PCR was performed to evaluate the expression level of T-bet. We also measured the concentration of the related cytokine (IFN-ɣ) in supernatants by ELISA.
The expression of T-bet as well as secretion of IFN-ɣ which is a Th1 related cytokine was significantly increased when a combination of both hormones were applied in case group compared to controls [Median: 84.04 (IQR: 77.32-177) and Median: 71.52 (IQR: 68.85-84.04) pg/ml respectively], however, treatment with these hormones alone did not show any significant effects.
We concluded that, treating PBMCs with estrogen and progesterone alone or in combination as an in vitro example of HRT, has stimulatory effect on Th1 cells’ behavior that may have a role in improving (sometimes worsening because of the complex role of CD8 cells) of allergic asthma symptoms. It is crucial to clarify the effect of these hormones on differentiated T helper cell population, which requires more studies to understand the effect of sex hormones on allergic asthma

In spite of the wide use, low proteome coverage and fuzzy patterns are the most important deterrents for the clinical applications of gel-based proteomic studies. So herein, we tried to increase the 2-dimentional proteome coverage of Hs578T breast cancer cells via investigating the efficacy of the three common techniques, usually used for interfering removal.
Hs578T cells were incubated in a lysis solution to obtain raw cell extracts. Cellular soups of each extraction were then pooled, homogenized, and aliquoted to be further treated by three different protein-specific purification methods including acetone, acetone-methanol, and trichloroacetic acid (TCA)-acetone, each in triplicates. All the purified protein pellets were then dissolved in a standard rehydration buffer solution, loaded into the 17-cm immobilized pH gradient (IPG) strips, and separated according to their isoelectric points. Proteins were then separated once more according to their molecular weights in an OFarrell separation system. Finally, by the visualization of the protein spots on the 2-dimentional profiles, quality and quantity of these 2-dimentional proteome patterns were then analyzed using the ImageMaster software.
Findings: The obtained proteome recovery yields and total protein counts for acetone, acetone-methanol, and trichloroacetic acid-acetone methods were 0.100 ± 0.001, 0.070 ± 0.002, and 0.120 ± 0.005 ng/cell, and 1299 ± 9, 1698 ± 14 and 1973 ± 17, respectively. The results represent data obtained from three independent experiments.
Trichloroacetic acid-acetone purification not only represented the highest recovery yield, suitable for expensive assays, but also showed the most suitable proteome coverage. So, the method is recommended for the comparative proteomic studies. However, the acetone-methanol procedure is more recommended for serological proteome analysis (SERPA); since it represents stronger protein spots which are more fitted to the immunoblotting procedure.

Tuberculin skin test is a common method for diagnosis of latent tuberculosis but it has a high rate of false positivity and false negativity. In Quantiferon test, the interferon gamma secreted by lymphocytes in response to Mycobacterium tuberculosis antigens is measured. This study aimed to determine the rate of agreement between quantiferon with skin tuberculin induration in the diagnosis of latent tuberculosis in elderly.
This study was carried out in 2015 on 10 elderly patients with confirmed active tuberculosis, 30 elderly patients with latent tuberculosis and 50 elderly individuals without any respiratory symptoms at Iran University of Medical Sciences, Tehran, Iran. The secretion of gamma interferon of specific T cells in peripheral blood was measured using Quantiferon test. In addition the tuberculin skin test was performed using standard methods and the skin induration was measured. By drawing the ROC curve, sensitivity, specificity, and under the curve area (AUC) was calculated interferon gamma release and tuberculin test. Results of patients and healthy people were compared with the Kruskal-Wallis test.
The sensitivity and specificity of gamma interferon release test for active tuberculosis in elderly patients was, 100% and 98% respectively (area under the curve 0.99) while the sensitivity and specificity of the tuberculin test was respectively 80% and 94% (area under the curve 0.89). Regarding to elderly patients with latent tuberculosis the sensitivity and specificity of interferon-gamma release test was 73% and 98%, respectively; and the sensitivity and specificity of tuberculin test in these patients was 36% and 94%, respectively.
Quantiferon is more sensitive and specific test for diagnosis of latent tuberculosis and application of this method is more reliable for diagnosis of latent tuberculosis infection in the elderly.

Aspergillus species has crucial role in respiratory allergy. This study was aimed to evaluate the pattern of IgE-mediated immune response to common aspergillus species including Aspergillus fumigatus, Aspergillus flavous, Aspergillus niger in type I aspergillus-hypersensitive patients’ sera.
The prepared protein extract of Aspergillus species were dialyzed and the protein concentrations were measured by Bradford method. The protein patterns were checked by SDS-PAGE. The allergic patients and controls were selected based on their clinical history and skin prick test (SPT) results. The cross-reactivity of allergens were determined by ELISA and Western-blotting using allergic patients’ and controls’ sera.
SDS-PAGE showed that the molecular weight of most of aspergillus proteins are between 11-100 kDa with typical bands between 46-100 kDa. ELISA showed that there is a significant difference between the serum-immunoreactivity of patients and negative controls for each of studied species. However, we did not find significant difference between the immunoreactivity of the patients with the studied fungal species. Western-blotting also detected various typical IgE-reactive proteins for each of aspergillus species.
The IgE-reactive pattern of aspergillus species shows a slight difference. The variation of the immunoreactivity of the aspergillus species could be due to the genetic variations, difference in the environmental predisposition with saprophyte molds or non-equal allergenic potency of the extracts.

It is stated that in the absence of angiogenesis, the tumoral tissue will not grow beyond 2 mm3. Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis and blockade of this process could be applied as a novel strategy for immunotherapy of cancer.
Peptide sequences of VEGF-A isoforms were retrieved from protein databases and aligned. Immunodominant epitopes were determined and the selected one was rechecked for dissimilarity with other human proteins. The selected conserved peptide sequence was synthesized and conjugated with Keyhole limpet hemocyanin (KLH). Then, it was applied for immunization of mice. The polyclonal anti-VEGF antibody titer was measured using an indirect peptide-enzyme-linked immunosorbent assay (ELISA) with a Bovine serum albumin (BSA)-conjugated peptide.
Findings: According to bioinformatic findings, the selected 41-aminoacid sequence did not show any similarity with other human proteins and revealed enough antigenicity to stimulate anti-tumor specific responses. A substantial increase of specific antibody titer was observed in vaccinated mice. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the BSA-conjugated peptide showed efficient coupling of the molecules. Optimization steps in ELISA procedures revealed that coating of microtiter plates with BSA-conjugated antigen provided more reproducible outcome than unconjugated peptide.
Our results reinforce the potential of KLH-conjugated peptides for immunization and production of specific polyclonal antibodies against VEGF-A. Production of high-titer antibodies against this antigen indicates that the designed peptide-vaccine could be used as a potential immunogen for stimulation of humoral immune system in animal model.

Metastasis is an advanced stage of cancer in which tumor cells are removed from the primary tumor and migrate to other areas of the body and develop a new mass by high replication. Animal models could be applied to evaluate metastasis along with a metastatic and appropriate cell line. Carcinoma 4T1 cell line is a metastatic cell line with excessive cell proliferation and invasion up to the liver, lungs, brain and lymph nodes.
By injection of about 5 ´ 105 4T1 cells to BALB/c mice, primary tumor was developed. Rate of tumor growth, survival and other morphologic symptoms were checked and recorded. To study the metastasis process, liver, lung and brain tissues were separated and the pathological status was studied following hematoxylin/eosin dye staining.
Findings: The primary tumor growth was increased significantly. The survival rate in mice with breast cancer was decreased to maximum in 7-8 weeks. Liver and lung tissues were metastatic with necrosis and infiltration of numerous cancer cells while metastasis was not observed in brain tissue.
In this model, high proliferation of cancer cells in mice was in association with a sharp decline in survival rate as well as lung and liver tissues metastasis. Metastasis to the brain cells might be accomplished by injection of more 4T1 cells and presumably occurs in a longer period.

Aims and This study was performed to evaluate the effect of lactobacillus acidophilus Bacteria (LAB) on chicken T cell subsets at ileum, bursa and cecal tonsil tissues. Thirty chickens were divided into three groups and fed with sterilized cow milk (M), or a mixture of M and LAB, kept as controls. Chickens from each group were sacrificed after 2 or 3 weeks and lymphoid tissues including ileum, bursa and cecal tonsil were removed and cryo preserved. The ratio of T cell subsets including CD4+, CD8+ and gamma delta T cells were studied by immune-histochemistry method. The ratio of CD4+ T cells was significantly higher in ileum of chicken after 2 weeks of probiotic intake. Moreover, this ratio was even higher than the ratio of these cells in the third week. In the cecal tonsil sections the percentage of gamma delta T cells was higher in milk fed chickens, after 3 weeks. The findings indicate that feeding of chickens with probiotics may alter the distribution of T cells in the lymphoid tissues. However, coincidental increase of CD4+ and gamma delta T cells after 2 and 3 weeks of treatment indicates that these effects are transient and could not have a permanent effect on avian mucosal immunity.

Leishmaniasis is a major infectious disease caused by protozoan parasites of the genusLeishmania.Despite of many efforts have been madeno effective vaccineagainst Leishmania infectionhas been approved yet.The major advantage of DNAvaccine isto induce the expression of antigens, which are unaltered in their protein structure and antigenicity. In this study, in order to increase immunity, thecandidate DNA vaccine has been supplemented with nano-adjuvant and its immunogenicity was tested on BALB/c rats. Considering other studies that have demonstratedLeishmania TSA protein is antigenic in both murine and human systems, in this study a new nanovaccinecontaining TSA recombinant plasmid and poly(methylmethacrylate) (PMMA) nanoparticles (as an adjuvant) was designed. After three intramuscular injection of nanovaccine (100 µg),the recombinant TSA protein(20 µg) was injected subcutaneously.3 weeks later,animal were infected by Leishmania major.Finally lymphocyte proliferation and cellular immune responses (IFN-γ,IL-4 prodution) were evaluated byusinigBrdu and ELISA methods. Theresultsof this study showed that the new nanovaccine was capable of inducing both cytokines secretion,but predominant Th1 immune response characterized by IFN-γ production compared to control groups. Results revealed that, current candidate nanovaccine has potency for future studies to prepare vaccine against Leishmania.

Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease, which requires specific diagnostic tests for it’s’ diagnosis. However specific and practical tests for its diagnosis are lacking. We evaluated the diagnostic accuracy of Anti-cyclic citrullinated peptide antibodies (Anti-CCP) in the diagnosis, prognosis and determination of the severity of RA. In a descriptive-cross sectional study Anti-CCP antibodies were determined in 140 serum samples: 82 from RA patients and 58 from controls, including samples of 30 healthy individuals and 28 patients with connective tissue diseases. Anti-CCP was detected by ELISA in the serum samples of patients, Rheumatoid factor of IgA, IgM and IgG classes was determined by ELISA and Anti-Nuclear Antibody was detected by Immunoflourescent method results were compared in the three groups. For comparison of differences between groups t (quantitative variables) and chi-square tests (qualititative variable) tests were used. Correlation between titer of IgM-RF and Anti-CCP was assessed by Pearson’s Correlation test. In addition, Receiver Operating Characteristics (ROC) analysis was carried out to compare sensitivity and specificity of various tests. For data analysis SPSS V. 15 was used. Mean concentration of Anti-CCP in RA patients that had Anti-CCP levels greater than normal was 95.1 IU/ml (range: 7.1-249 IU/ml). While mean concentration of Anti-CCP in RA patients that had Anti-CCP levels less than normal was 2.1 IU/m (range: 1.2-4.9 IU/ml).Controls had a mean Anti-CCP level of 7.35 IU/ml (range: 2.44-13.26 IU/ml) Diagnostic sensitivity and specificity of RF was reported as 81.7% and 44.6% respectively in the patients. As a screening method for rheumatoid arthritis IgM-RF, Anti-CCP antibody assays are superior to other RF isotypes assessments. Anti-CCP proved to be a powerful and specific diagnostic tool, especially in patients with undiagnosed rheumatoid arthritis.

Worldwide increasing number of allergic disorders is one of the most important health challenges, and achieving safe prophylactic and therapeutic procedures is the main aim of the investigators. CpG oligodeoxynucleotides (CpG ODN) which modulate the immune responses and change the cytokine patterns by severalmechanisms are among these procedures. In this experimental study we investigated the effects of CpG motif on immune responses to Chenopodium album (CH.a), a common allergenic plant in Iran, in a murine model of asthma. We used CH. a pollen extract with CpG to sensitize the mice and compared a number of immunologic parameters such as interferon gamma (IFN- 􀇄) and IgE levels between case and control groups. To do this, IFN-􀇄 and IgE levels were measured in splenocyte and lung culture supernatants. Histological studies were also conducted to identify inflammatory cells in lung airways. ANOVA test was used to analyze the data. The study showed although CpG ODN cannot reduce IgE levels to the normal range, they can lower IgE levels by 80% and have a potent influence to augment both systemic and local levels of IFN-􀇄 as a Th1 activity marker (P<0.01) The results of the study have consistency with other reports and confirm the benefits of CpG motifs in reducing lung inflammatory responses. Since CpG components have potency to shift immune activity from Th2 to Th1 responses, it seems that co-administration of CpG/antigen can modulate inflammatory responses to different allergenic antigens.

Evaluation of Matrix Metaloproteinase 2 (MMP2) Activity in Contact Nickel Dermatitis Using Zymoanalysis in Comparison to Normal Individuals. R. Falak MSc*M.R. Khoramizadeh MD**M. Pezeshki MD*** P. Mansoori MD**** * Master of Seience of Immunology Iran University of Medical Seiences ** Associate Professor of Biotechnology Faculty of Health Tehran University of Medical Sciences *** Associate Professor of Immunology Faculty of Health Tehran University of Medical Sciences **** Professor of Dermatology Imam Khomeini Hospital Tehran University of Medical Sciences Background and objective Matrix Metaloproteinases (MMPs) produced mainly by fibroblasts play a major role in wound healing processes. This study aimed to investigate the MMP2 activity in dermal fibroblasts found in chronic contact nickel dermatitis lesions. Methods In order to study the role of MMP2 in contact nickel dermatitis fibroblast from patients and healthy individuals were cultured based on ex-plantation of skin. MMP2 activity was measured in fibroblast culture media using zymoanalysis. Zymograms were then analyzed by quantitative densitometry. MTT assay was also used to evaluate and compare proliferation capacity of fibroblasts in both cell cultures. Results The mean MMP2 activity 6-8 days after ex-plantation was significantly higher in patients fibroblasts than in normal individuals(170±9.2 and 134.3±5.9 in patients and normals respectively.

The immune system is quite capable to combat tumors and many immunological parameters including cytokines such as IL-12 & IFN-γ play major roles in this regard. IL-12 is also the major cytokine responsible for the differentiation of TH1 cells, which are potent producers of IFN-γ, IFN-γ in turn has a powerful enhancing effect on the ability of phagocytes to produce IL-12 as well as having an important role in cellular immune response. In this study the serum concentration of IL-12 & IFN-γ and percentage of IFN-γ producing cells(CD4+, CD8+, NK cells) in metastatic & nonmetastatic breast cancer patients and healthy adults was evaluated, with the aim of finding out the possible correlation between cytokine levels with disease stage and progression. Since cytokines are produced by all of these cells, cell enumeration may help to find out whether changes in cytokine levels is due to change in the cell number or their function has been altered during disease progression. Patients and Whole blood samples were taken from 50 breast cancer patients prior to therapeutic manipulation who were admitted to Dr. Shariaty & Day general hospitals. Also 26 healthy people were selected as control and blood was taken similarly. According to disease stage patients were classified into non-metastatic(stage I, II) and metastatic(stage III, IV) groups. 30 patients were non metastatic and 20 patients were in metastatic stages respectively. Serum cytokines level(IFN-γ, IL-12) was measured by ELISA. Lymphocyte subpopulation percentage was measured by flow-cytometry using bichromatic specific antibodies(anti-CD4+/CD8+, anti-CD3/CD16+CD56). The research protocol is designed as a descriptive, comparative cross sectional study. The parametric findings were analysed by one way Anova test and the nonparametric data were analysed using Mann whitney and Kruskal Wallis statistical tests. IL-12 level was increased significantly in metastatic group compared to controls(P=0.017), while IFN-γ levels were in the same range as controls. CD4+ lymphocyte percentage in nonmetastatic(P=0.022) and metastatic groups(P=0.037) was decreased significantly compared to control, but there was no significant difference in CD8+ and NK cell numbers. Despite the decrease in percentage of CD4+ lymphocytes in patients(due to activation of compensative hemostatic system and increase in IL-12), there was no change in IFN- level. It seems that increase in serum IL-12 levels correlates with disease progression. However serum IFN-γ level has no effect on disease progression and as a whole no prominent failure was recorded in the cellular immune response of brease cancer patients as compared to normal individuals.

Nasal polyp is a common finding in patients referring to ENT specialists. Also, some specialists believe that there are patients with nasal polyp and some evidence of fungi. The present study was undertaken to describe coincidence of nasal polyp and fungi in patients referred to ENT ward of Rasoul-e-Akram Hospital in Tehran. Patients & 118 patients with nasal polyp(62.7% male and 37.3% female mean age 35 years, ranged 12-70) were investigated in a cross-sectional study. 52.5% of the patients had high total serum IgE level. Direct examination and culture for fungi was positive in 32.5% and negative 36.4% of the patients. The most common isolated fungus was aspergillus(50.9%). Other fungi were candida(34.5%), penicillium (7.3%), alternaria (5.5%) and curvularia (1.8%). Mean age of patients with fungal infection was higher than patients with negative fungal culture result (40y vs. 35y P=0.06). Patients with high serum IgE level were more infected with fungi (49% vs. 14% P<0.001). Results showed that in nasal polyposis, a special attention must be paid to patients with high serum IgE and fungal infection.However, most of our patients had chronic sinusitis as well as polyps in their medical history which may affect our results. Therefore, further studies on patients suffering from nasal polyps and those cases with fungal sinusitis is suggested.